Estudio de la utilidad y mecanismos de acción de las células madre adultas del tejido adiposo para el tratamiento del rechazo tras trasplante corneal alogénico

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 18-12-2015En el presente estudio se investiga la utilidad de las células madre mesenquimales adultas derivadas de tejido adiposo (AD-MSC) en modelos de trasplante corneal alogénico de riesgo normal y de alto riesgo en conejos. Se pretende conseguir una prevención del rechazo y un aumento de la vida media del injerto mediante las propiedades inmunosupresoras atribuidas a las AD-MSC, así como establecer modelos animales más parecidos a lo que se encuentra en la práctica clínica en humano y por tanto más relevantes que los descritos hasta el momento. En contra de lo esperado, en el modelo de riesgo normal, establecido usando dos razas diferentes de conejo, la aplicación local de AD-MSC durante la cirugía no aumentó el tiempo de supervivencia del trasplante sino que lo disminuyó, aumentando la vascularización del lecho corneal, así como los signos de inflamación, concretados en un aumento del edema y la infiltración leucocitaria. En el modelo de alto riesgo establecido mediante un proceso de vascularización previa de la córnea de conejo, la aplicación sistémica de AD-MSC de conejo en varios momentos antes, durante y después de la cirugía también disminuyó el tiempo de supervivencia del trasplante, aumentando la vascularización y el edema corneal. Estos resultados pueden ser explicados por el carácter proinflamatorio mostrado por las AD-MSC in vitro. Estas células expresan y secretan de forma constitutiva varias citoquinas proinflamatorias como IL-6 e IL-8. El efecto de estas citoquinas parece sobrepasar el de factores inmunosupresores como IDO y NO también secretados por las AD-MSC en este estudio. Además, estas células presentan un fenotipo de célula presentadora de antígeno por la expresión de moléculas coseñalizadoras como CD40 y CD80. Por último, las AD-MSC expresan citoquinas proangiogénicas como VEGF-A y TGF-β relacionadas con un aumento de la neovascularización. Todos estos datos sugieren que las AD-MSC no serían adecuadas para su uso en tejidos avasculares e inmunoprivilegiados como la córnea, ya que previenen el mantenimiento de la homeostasis del tejidoThe effect of adipose-derived mesenchymal stem cells (AD-MSC) into rabbit models of normal or high risk corneal allograft transplantation was investigated. Our aims were to prevent transplant rejection and to increase the time of graft survival mediated by the described immunosuppressive properties of AD-MSC, and to establish more similar corneal graft models to the clinical practice in humans, and thus more relevant than the previously reported rodent models. The normal risk corneal graft model was established by using two different rabbit strains. In contrast to our expectations, the local administration of AD-MSC during surgery worsen the outcome of the transplantation, increasing vascularization of the corneal bed and inducing signs of inflammation such as edema and leukocyte infiltration. This resulted in a shorter graft survival. In the high risk corneal graft model, immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery. Systemic administration of rabbit AD-MSC at various time points also led to a shorter survival time. Rabbit AD-MSC increased vascularization and edema in these corneal grafts. Our results may be explained by the proinflammatory phenotype shown by AD-MSC in vitro. These cells constitutively express and secrete proinflammatory cytokines and chemokines such as IL-6 and IL-8. The effect of those cytokines seems to overcome that of immunosuppressive factors such as IDO and NO also secreted by MSC in the present graft model. In addition, these cells present an antigen presenting cell phenotype by the expression of cosignaling molecules CD40 and CD80. Moreover, AD-MSC express proangiogenic factors such as VEGF-A and TGF-β related to neovascularization processes. All these data suggests that AD-MSC may not be appropriate for their use in avascular and immuneprivileged sites as the cornea, as they prevent the maintenance of the tissue homeostasi

Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD

Pre-pandemic Alzheimer Disease Biomarkers and Anxious-Depressive Symptoms During the COVID-19 Confinement in Cognitively Unimpaired Adults

BACKGROUND AND OBJECTIVES: Increased anxious-depressive symptomatology is observed in the preclinical stage of Alzheimer's disease (AD), which may accelerate disease progression. We investigated whether amyloid-β, cortical thickness in medial temporal lobe structures , neuroinflammation and sociodemographic factors were associated with greater anxious-depressive symptoms during the COVID-19 confinement. METHODS: This retrospective observational study included cognitively unimpaired older adults from the ALFA (Alzheimer and FAmilies) cohort, the majority with a family history of sporadic AD. Participants performed the Hospital Anxiety and Depression Scale (HADS) during the COVID-19 confinement. A subset had available retrospective (on average: 2.4 years before) HADS assessment, amyloid [18F] flutemetamol PET and structural MRI scans and CSF markers of neuroinflammation (interleukin-6 [IL-6], triggering receptor expressed on myeloid cells 2 and glial fibrillary acidic protein levels). We performed multivariable linear regression models to investigate the associations of pre-pandemic AD-related biomarkers and sociodemographic factors with HADS scores during the confinement. We further performed an analysis of covariance in order to adjust by participants' pre-pandemic anxiety-depression levels . Finally, we explored the role of stress and lifestyle changes (sleep patterns, eating, drinking, smoking habits, and medication use) on the tested associations and performed sex-stratified analyses. RESULTS: We included 921 (254 with AD biomarkers) participants. Amyloid-β positivity (B=3.73; 95%CI=1.1 to 6.36; p=.006), caregiving (B=1.37; 95%CI=0.24 to 2.5; p=.018), sex (women: B=1.95; 95%CI=1.1 to 2.79; p<.001), younger age (B=-0.12; 95%CI=-0.18 to -0.052; p<.001) and lower education (B=-0.16; 95%CI=-0.28 to -0.042; p=.008) were associated with greater anxious-depressive symptoms during the confinement. Considering pre-pandemic anxiety-depression levels, we further observed an association between lower levels of CSF IL-6 (B=-5.11; 95%CI=-10.1 to -0.13; p=.044) and greater HADS scores. The results were independent of stress-related variables and lifestyle changes. Stratified analysis revealed that the associations were mainly driven by women. DISCUSSION: Our results link AD-related pathophysiology and neuroinflammation with greater anxious-depressive symptomatology during the COVID-19-related confinement, notably in women. AD pathophysiology may increase neuropsychiatric symptomatology in response to stressors. This association may imply a worse clinical prognosis in people at risk for AD after the pandemic, and thus deserves to be considered by clinicians. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier NCT02485730

Genetically predicted telomere length and Alzheimer’s disease endophenotypes: a Mendelian randomization study

Telomere length (TL) is associated with biological aging, consequently influencing the risk of age-related diseases such as Alzheimer's disease (AD). We aimed to evaluate the potential causal role of TL in AD endophenotypes (i.e., cognitive performance, N = 2233; brain age and AD-related signatures, N = 1134; and cerebrospinal fluid biomarkers (CSF) of AD and neurodegeneration, N = 304) through a Mendelian randomization (MR) analysis. Our analysis was conducted in the context of the ALFA (ALzheimer and FAmilies) study, a population of cognitively healthy individuals at risk of AD. A total of 20 single nucleotide polymorphisms associated with TL were used to determine the effect of TL on AD endophenotypes. Analyses were adjusted by age, sex, and years of education. Stratified analyses by APOE-epsilon 4 status and polygenic risk score of AD were conducted. MR analysis revealed significant associations between genetically predicted longer TL and lower levels of CSF A beta and higher levels of CSF NfL only in APOE-epsilon 4 non-carriers. Moreover, inheriting longer TL was associated with greater cortical thickness in age and AD-related brain signatures and lower levels of CSF p-tau among individuals at a high genetic predisposition to AD. Further observational analyses are warranted to better understand these associations

Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome.

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice

Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium.

ObjectiveWe aimed to investigate the functionality of human decellularized stromal laminas seeded with cultured human corneal endothelial cells as a tissue engineered endothelial graft (TEEK) construct to perform endothelial keratoplasty in an animal model of corneal endothelial damage.MethodsEngineered corneal endothelial grafts were constructed by seeding cultured human corneal endothelial cell (hCEC) suspensions onto decellularized human corneal stromal laminas with various coatings. The functionality and survival of these grafts with cultured hCECs was examined in a rabbit model of corneal endothelial damage after central descemetorhexis. Rabbits received laminas with and without hCECs (TEEK and control group, respectively).ResultshCEC seeding over fibronectin-coated laminas provided an optimal and consistent endothelial cell count density and polygonal shape on the decellularized laminas, showing active pump fuction. Surgery was performed uneventfully as standard Descemet stripping automated endothelial keratoplasty (DSAEK). Corneal transparency gradually recovered in the TEEK group, whereas haze and edema persisted for up to 4 weeks in the controls. Histologic examination showed endothelial cells of human origin covering the posterior surface of the graft in the TEEK group.ConclusionsGrafting of decellularized stroma carriers re-surfaced with human corneal endothelial cells ex vivo can be a readily translatable method to improve visual quality in corneal endothelial diseases

Representative photographs of histological sections of transplanted corneas.

<p>A) sham group; B) human AD-MSCs; C) activated human AD-MSCs; and D) rabbit AD-MSCs. Note the intense edema causing thickening of the corneal stroma (Str) in all MSC treated corneas, as well as leukocyte infiltration in the stroma. Epi: corneal epithelium; End: corneal endothelium. Hematoxylin and eosin staining. E) Corneal thickness measurements in the transplanted corneas. Stars indicate statistical significance at the p≤0.05 level. F) CD45 leukocyte infiltration measurements. Stars indicate statistical significance at the p≤0.05 level. G) CD45 immunohistochemistry in a transplanted cornea with sham treatment showing no positive cells. H) CD45 immunohistochemistry in a AD-MSC treated transplanted cornea. Positive cells are labeled in brown (arrows).</p

A) Survival curve of the corneal grafts of the high-risk model of corneal transplant.

<p>Graft survival was significantly shortened by intravenous injection of rabbit MSCs (rAD-MSCs) compared with vehicle injection (sham group), p = .085. B,C) Rejection index score (haze, edema, and neovascularization) comparison between groups. Different colors show different rejection index scores in the surviving grafts at any time point.</p

AD-MSC characterization.

<p>A-D: Fluorescence-activated cell sorting of a sample of the stromal vascular fraction of human adipose tissue for markers CD45, CD34, CD90 and CD105 (open lines) with respect to autofluorescence (shadow lines). Percentages of each population are given within the graph for each marker. E: Phase contrast of cultured living AD-MSC cells. Observe the normal fusiform morphology. F: In vitro osteogenesis of the human AD-MSC, revealed by alkaline phosphatase staining with Fast Red. G: In vitro chondrogenesis of the human AD-MSC cultured using the micromass technique and revealed by Alcian blue. H: In vitro adipogenesis of the human AD-MSC, revealed by Oil red staining in red.</p

Representative photographs of transplanted corneas at the time of rejection.

<p>A) sham group; B) human AD-MSCs; C) activated human AD-MSCs; D) rabbit AD-MSCs after transplantation; E) unrejected cornea of one of the sham group rabbits; F) unrejected cornea of one of the MSC treated rabbits. Magnification 2x.</p